Abstract: :
Purpose: The established retinal pigment epithelial (RPE) cellline ARPE–19 is commonly used to study RPE cell behavior.While this cell line has the potential to develop differentiatedproperties, the optimal global gene expression changes underdifferent culture conditions have not been established. Thepurpose of this work was to determine the proximity of expressionprofiles by RPE cells grown under different culture conditionsto native RPE.Methods: Morphologically normal RPE cells overlying nonthickenedBruch’s membrane (n=5000 cells) from 10 eyes (45–95yrs) were laser capture microdissected (LCM).ARPE–19 cellswere grown in subconfluent, visually confluent, and high confluentfor 2.5 months with or without serum. Total RNA was used tosynthesize first and second strand 33P–dATP and 33P–dCTPcDNA probes which were hybridized to a ResGen array (5353 genes),and the signal was visualized by phosphorimager analysis. TheTIFF images were analyzed by ResGen’s Pathways 3 software.Experiments were repeated twice (n=3) for each condition. Thedata were normalized to a mean signal reference array, and subsequentlyanalyzed using Cluster/Treeview and SAM. Real time PCR was usedto confirm differential expression of selected genes.Results: The expression profiles of ARPE–19 cells weregrown under 5 culture conditions were compared to morphologicallynormal native RPE cells that were laser capture microdissectedfrom 5 eyes. While 78% of genes on the array were expressedby native RPE, 45.3–47.7% of genes were expressed by ARPE–19cells, depending on culture condition. The most abundant geneswere commonly expressed by native and cultured cells, but significantdifferences in low abundance genes were seen between nativeand cultured RPE. Unsupervised and supervised cluster analysisshowed that confluent and differentiated, serum withdrawn culturesclustered closest to native RPE, and that serum segregated culturedcells away from native RPE. The number of differentially expressedgenes, function of differentially expressed genes, and profileof expressed and unexpressed genes demonstrate significant differencesbetween native and ARPE–19 cells with the culture conditionschosen for this study.Conclusions: While cultured RPE cells and in particular ARPE–19cells, have significant value for studying RPE behavior, investigatorsmust be aware of how culture conditions can influence the mRNAphenotype of the cell.